Methods of producing recombinant insulin from novel fused proteins

ABSTRACT

This invention relates to a DNA encoding a fusion protein of formula (I)
 
[Y]-[X1]-[B-chain]-[X2]-[Linker]-[X3]-[A-chain]  (I)
 
wherein
         Y is a leader peptide sequence for the expression and secretion of the protein, comprising at least one amino acid residue;   X1 is an amino acid sequence which is cleavable enzymatically or chemically;   B-chain is the amino acid sequence of insulin B chain;   X2 is an amino acid sequence which is cleavable enzymatically;   Linker is a linker sequence comprising at least one amino acid residue;   X3 is an amino acid sequence which is cleavable enzymatically; and   A- chain is the amino acid sequence of insulin A chain,
 
and to a method for producing insulin using an expression system containing the DNA in high efficiency and high yield.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP00/02736 which has an Internationalfiling date of Apr. 26, 2000, which designated the United States ofAmerica.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a DNA encoding a novel fusion proteinwhich is used for preparing recombinant insulin. More specifically, thepresent invention relates to the use of the DNA for the preparation ofinsulin from the fusion protein, which is obtained by the expression ofthe DNA, through the action of thrombin and carboxypeptidase B.

2. Background Art

Insulin is a hormone secreted by the B cells of the islet of Langerhansin the pancreas when an animal ingests food, which is the most importanthormone for storage or use of sugars, amino acids and fatty acids andfor maintaining the blood sugar homeostasis. Although blood sugar,namely glucose in blood, is an essential energy source for a livingbody, if the blood sugar homeostasis is not maintained, then seriousconditions may develop. Increased blood sugar level causes the excretionof sugar in the urine, resulting in loss of glucose, i.e. onset of aso-called diabetes. If this condition continues for long periods,complications can develop in the tissues of a living body. On the otherhand, decreased blood sugar level leads to an insufficient supply of theenergy source, resulting in imperilment of life. Homeostasis of theblood sugar level is maintained by balancing factors that act toincrease the blood sugar level (e.g., glucagon, growth hormone,cortisol, catecholamine) with factors that act to decrease the bloodsugar level. Insulin is the only hormone which can decrease the bloodsugar level. Hence, the reduction in secretion functions resulted fromsome causes and, as a consequence, insufficient supply of insulin caninduce insulin-dependent diabetes mellitus (IDDM). For patientssuffering from such a disease, insulin is an indispensable drug.

Human insulin is a polypeptide comprising an A chain with 21 amino acidsand a B chain with 30 amino acids, which has one intrachain disulfidebond in the A chain and two disulfide bonds which link between the Achain and the B chain. Insulin is initially biosynthesized as“preproinsulin” on ribosomes in B cells of the islets of Langerhans ofthe pancreas. Preproinsulin is a linear molecule comprising a signalpeptide with 24 amino acids (SP), a B chain (B), a C-peptide with 31amino acids (C) and an A chain (A) linked in the order as represented bythe formula “SP-B-C-A”. Upon transport to the endoplasmic reticulum, thesignal peptide is cleaved out from the preproinsulin to produce“proinsulin (B-C-A)”. Proinsulin forms disulfide bonds in theendoplasmic reticulum, thereby taking on a three-dimensional structure.Proinsulin is cleaved with a prohormone-converting enzyme PC1/3 at theB-C junction and then cleaved with a converting enzyme PC2 at the C-Ajunction. Finally, N-terminal two basic amino acid residues of theC-peptide, which remain at the C-terminus of the B-chain when cleavedwith PC1/3, are cut out with carboxypeptidase H. In this manner, insulinis produced.

Methods for producing therapeutic insulin have been initially developedusing extracts from the pancreas of animals such as bovine and pig.However, human insulin is different in amino acid composition frombovine insulin (at two positions in A chain and one position in B chain)and porcine insulin (at one position in B chain). Therefore, adverseeffects (e.g., allergy) are inevitable in the use of bovine or porcineinsulin in human bodies. Methods for semi-synthesis of human insulinfrom porcine insulin have been developed which utilize thetranspeptidation reaction with trypsin. However, recombinant insulinproduced by genetic recombinant techniques has currently gone mainstreamdue to its low production cost and good production efficiency.

For the production of recombinant insulin, a number of methods have beendeveloped. For example, the method developed by Eli Lilly Corp. isknown, which method comprises expressing A chain and B chain separatelyusing Escherichia coli; and mixing the A chain and the B chain in vitroto form the disulfide bridges, thereby linking them via the disulfidebonds (JP-B- 63-18960). This method, however, is poor in productionefficiency. Then, Eli Lilly Corp. has developed an improved method whichcomprises expressing proinsulin; forming the disulfide bonds in vitro;and then cleaving out the C-peptide from the product with trypsin andcarboxypeptidase B, thereby producing insulin (JP-B-1-48278 and JapanesePatent No. 2634176).

Another method was developed by Novo Nordisk Corp., which methodcomprises expressing miniproinsulin comprising a B chain and an A chainlinked via two basic amino acid residues, in yeast; and then treatingthe miniproinsulin with trypsin in vitro, thereby producing insulin(JP-B-7-121226 and JP-B-8-8871, and Japanese Patent No.2553326). Thismethod has such advantages that the disulfide bonds are formed duringthe expression and secretion of the miniproinsulin and that theminiproinsulin can be isolated and purified readily because it issecreted into a cultured medium.

Development of new recombinant insulin-production methods has beencontinued positively. Hoechst Corporation developed a method comprisingexpressing a new-type insulin derivative or preproinsulin in E. coli;forming the disulfide bonds in vitro; and then treating the product withlysylendopeptidase or clostripain/carboxypeptidase B, thereby producinginsulin (JP-A-2-195896, JP-A-2-225498, JP-A-2-233698, JP-A-3-169895,JP-A4-258296, JP-A-6-228191, and JP-A-7-265092). Recently, a method hasbeen developed by BIO-TECHNOLOGY GENERAL CORPORATION, in which a fusionprotein comprising superoxide dismutase (SOD) linked with proinsulin isexpressed in E. coli to increase both the expression efficiency and thedisulfide bond-forming efficiency, and proinsulin is converted intoinsulin with trypsin and carboxypeptidase B (WO 96/20724). Thus, thereare a number of approaches for recombinant insulin production, andfurther improvement has been made in expression efficiency, disulfidebond-forming efficiency and conversion into insulin.

As the hosts for the production of recombinant proteins, a wide varietyof hosts have been used including microorganisms, animals and plants.Among them, microorganisms are most frequently used due to theireasy-to-handle property and good applicability for industrial use, andEscherichia coil and yeast are especially known. Recently, an expressionsystem with Bacillus brevis has been known for recombinant proteins (seeJapanese Patent No. 2082727; JP-A-62-201583; Yamagata, H. et al., J.Bacteriol. 169:1239-1245, 1987; Juzo Udaka, Nihon Nogei Kagaku-shi 61,669-676, 1987; Takao, M. et al., Appi. Microbiol. Biotechnol. 30:75-80,1989; Yamagata, H. et al., Proc. Natl. Acad. Sci. USA 86:3589-3593,1989).

The object of the present invention is to develop an expression systemand a production method for insulin which have a high yield and aproduction efficiency equal to or better than those of the existingrecombinant insulin production systems. That is, the object of thepresent invention is to develop a novel method for converting an insulinprecursor into insulin, an environment where the disulfide bondsnecessary for insulin activity can be formed, and an expression systemwith high yield.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a DNA encodinga fusion protein of formula (1):[Y]-[X1]-[B-chain]-[X2]-[Linker]-[X3]-[A-chain]  (I)wherein

-   -   Y is a leader peptide sequence for expression and secretion of        the protein, comprising at least one amino acid residue;    -   X1 is an amino acid sequence which is cleavable enzymatically or        chemically;    -   B-chain is the amino acid sequence of insulin B chain;    -   X2 is an amino acid sequence which is cleavable enzymatically;    -   Linker is a linker sequence comprising at least one amino acid        residue;    -   X3 is an amino acid sequence which is cleavable enzymatically;        and    -   A-chain is the amino acid sequence of insulin A chain, and

wherein the Y, X1, B-chain, X2, Linker, X3 and A-chain are ligated inthe order indicated in formula (I).

In another aspect of the present invention, the present invention isprovide a DNA encoding a fusion protein of formula (II):[B-chain]-[X2]-[Linker]-[X3]-[A-chain]  (II)wherein:

-   -   B-chain is the amino acid sequence of insulin B chain;    -   X2 is an amino acid sequence which is cleavable enzymatically;    -   Linker is a linker sequence comprising at least one amino acid        residue;    -   X3 is an amino acid sequence which is cleavable enzymatically;        and    -   A-chain is the amino acid sequence of insulin A chain, and

wherein the B-chain, X2, Linker, X3 and A-chain are ligated in the orderindicated in formula (II).

In one embodiment of the present invention, the amino acid sequence X1,X2 or X3, which is used for enzymatic cleavage of a fusion protein, is asequence cleavable with thrombin. For example, the amino acid sequencewhich is cleavable with thrombin is the following sequence:

X1=GlySerLeuGlnProArg (SEQ ID NO:1);

X2=ArgGlyHisArgPro (SEQ ID NO:2); or

X3=ProArg.

In another embodiment of the present invention, the linker sequence hasthe following amino acid sequence:

GluAlaGluAspLeuGlnValGlyGlnValGluLeuGlyGlyGlyProGlyAlaGlySer

LeuGlnProLeuAlaLeuGluGlySerLeuGln (SEQ ID NO:3).

In still another embodiment of the present invention, the leader peptidesequence may comprise N-terminal 9 amino acid residues (i.e., the aminoacid positions 1 to 9) of the protein MWP which is one of the cell wallproteins (CWPS) derived from a bacterium belonging to the genusBacillus. In this case, the DNA may comprise a CWP signal peptideattached at the 5′ end of the DNA.

An example of the DNA of the present invention is a DNA comprising anucleotide sequence encoding an amino acid sequence shown in SEQ IDNO:21. More specifically, the DNA comprises a nucleotide sequence shownin SEQ ID NO:20.

In another aspect of the present invention, there is provided a DNAcomprising a DNA sequence which comprises a promoter region required forthe expression of a recombinant protein in a prokaryote or eukaryote,the DNA sequence being attached at the 5′ end of the DNA defined above.

In an embodiment of the present invention, the DNA sequence whichcomprises a promoter region is derived from a bacterium belonging to thegenus Bacillus, and is preferably derived from the CWP from a bacteriumbelonging to the genus Bacillus.

In another aspect of the present invention, there is provided a vectorcontaining the DNA defined above.

In still another aspect of the present invention, there is provided ahost cell transformed with the vector. The host cell is preferably abacterium belonging to the genus Bacillus, such as Bacillus brevis.

In still another aspect of the present invention, there is provided amethod for producing insulin, wherein the method comprises:

-   -   culturing the host cell or bacterium in a culture medium, to        express a fusion protein encoded by a DNA of interest in the        host cell or bacterium;    -   collecting the fusion protein; and    -   subjecting the fusion protein to an enzymatic cleavage treatment        to isolate insulin.

In this aspect, an example of the DNA is a DNA comprising a nucleotidesequence shown in SEQ ID NO:21, and an example of the fusion protein isa protein comprising an amino acid sequence shown in SEQ ID NO:22.

In the method, the expressed fusion protein may be separated andpurified from the host cell or the bacteria or the cultured medium.According to an embodiment of the present invention, the enzymaticcleavage may be performed with thrombin and carboxypeptidase B.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the conversion of a fusion proteinMWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain into insulin.

FIG. 2 is the amino acid sequence of a fusion proteinMWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain and the nucleotide sequenceencoding the same.

FIG. 3 is the schematic diagram depicting a process of introducing afusion DNA into a Bacillus brevis expression vector (pNU211R2L5).

FIG. 4 is the photograph of the electrophoresis of the media afterculture of transformants: lane 1, a marker peptide; lane 2, a negativecontrol (i.e., a transformant with only plasmid pNU211R2L5 and without aforeign protein); and lane 3, a transformantMWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain.

FIG. 5 shows the chromatogram of the fusion proteinMWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain isolated and purified by XLchromatography.

FIG. 6 shows the chromatogram of the fusion proteinMWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain isolated and purified byHPLC.

FIG. 7 shows the peptide mapping of ITOHAM insulin (the presentinvention) and Novolin Novolin 40, which is a commercially availableinsulin, sold by Novo Nordisk Pharma).

FIG. 8 shows the elution patterns of ITOHAM insulin (the presentinvention) and Novolin.

FIG. 9 shows the time courses of the plasma glucose levels afteradministration of ITOHAM insulin (the present invention) and Novolin.

FIG. 10 shows the time courses of the plasma insulin levels afteradministration of ITOHAM insulin (the present invention) and Novolin.

DETAILED DESCRIPTION

The DNA of the present invention has the structure represented by theabove formula (I) or (It). The DNA of formula (I) comprises a leaderpeptide sequence for expression and secretion of a fusion protein ofinterest, comprising at least one amino acid residue (Y); an amino acidsequence which is cleavable enzymatically or chemically (X1); a aminoacid sequence of insulin B chain (B-chain); an amino acid sequence whichis cleavable enzymatically (X2); a linker sequence comprising at leastone amino acid residue (Linker); an amino acid sequence which iscleavable enzymatically (X3); and an amino acid sequence of insulin Achain (A-chain), wherein the Y, X1, B-chain, X2, Linker, X3, and A-chainare ligated in this order. The DNA of formula (II) comprises a aminoacid sequence of insulin B chain (B-chain); an amino acid sequence whichis cleavable enzymatically (X2); a linker sequence comprising at leastone amino acid residue (Linker); an amino acid sequence which iscleavable enzymatically (X3); and an amino acid sequence of insulin Achain (A-chain), wherein the B-chain, X2, Linker, X3, and A-chain areligated in this order. The presence of the leader peptide sequenceenables to secrete the expression product outside the host cell, whilethe absence thereof provides the accumulation of the expression productwithin the host cell.

In the examples described below, for establishment of a method forconverting the fusion protein into insulin with thrombin andcarboxypeptidase B, a novel modified proinsulin is designed in whichthrombin-cleavage sites are provided between the insulin B chain and thelinker peptide and between the linker peptide and the insulin A chain.The modified proinsulin is then linked at its N-terminus, with theN-terminal 9-amino acids of a cell wall protein (CWP) from Bacillusbrevis as a leader peptide, and a third thrombin-cleavage site whichenables the cleavage of the modified proinsulin from the leader peptideis further linked immediately after the leader peptide, therebyproviding an environment for forming disulfide bonds and enabling theexpression of the modified proinsulin in Bacillus brevis. In thismanner, the linear artificial fusion protein which comprises a leaderpeptide, a thrombin-cleavage site, insulin B chain, a thrombin-cleavagesite, a linker peptide, a thrombin-cleavage site, and insulin A chain inthe order, can be designed. At first, a DNA encoding the correspondingfusion protein was prepared and inserted into a suitable expressionvector. The vector is then introduced into a suitable host cell. Thetransformed host cell is cultured to express the DNA, thereby producingthe fusion protein. The fusion protein is then enzymatically cleavedwith thrombin and carboxypeptidase B. Thus, insulin having the desiredprimary structure and biological activity, which are identical withthose of the naturally occurring insulin, can be produced.

The present invention will be illustrated in more detail hereinafter.

The leader peptide (Y), which comprises at least one amino acid residuerequired for the expression of a protein of interest, includes the knownMBP (Maina, C.V. et al., Gene 74:365-373, 1988), GST (Smith, D. B., etal., Gene 67:31-40, 1988), TRX (La Vallie, E. R. et al., Bio/Technology11:187-193, 1993), DsbA (Collins-Racie, L.A. et al., Bio/Technology13:982-987, 1995) and LamB (Benson, S. A. et al., Cell 32:1325-1335,1983), which are derived from E. coli; and α factor derived from yeast(Brake, A. J., Yeast Genetic Engineering, p269-280, 1989). Inparticular, the leader sequence is often required, for E. coli, when aprotein of interest is secreted into the periplasm or, for yeast, whenthe protein is secreted into the culture medium. According to anembodiment of the present invention, a preferred leader peptidecomprises the N-terminal 9 amino acids of a CWP from a bacteriumbelonging to the genus Bacillus (hereinafter, sometimes referred to as“Bacillus bacterium”). The CWP useful for the present inventionincludes, but is not limited to, those derived from Bacillus brevisstrain 47 (FERM P-7224; JP-A- 60-58074 and JP-A-62-201589), strain HPD31 (FERM BP-1087; 1P-A- 4-278091). Specifically, the following sequencesmay be used (where the brackets show references).

MWPmp9: AlaGluGluAlaAlaThrThrThrAla

-   -   (SEQ ID NO:4; J. Bacteriol., 169:1239-1245, 1989)

OWPmp9: AlaProLysAspGlylleTyrIleGly

-   -   (SEQ ID NO:5; J. Bacteriol., 170:176-186, 1988)

HWPmp9: AlaGluAspThrThrThrAlaProLys

-   -   (SEQ ID NO:6; J. Bacteriol., 172:1312-1320, 1990)

The number of amino acid residues from the N-termninus of the CWP is notnecessary to be 9, as long as the fusion protein can be expressed. Forexample, a sequence comprising N-terminal 1-50 amino acid residues ofthe CWP from a Bacillus bacterium may be used. The leader peptide is notalways be needed if the fusion protein portion behind insulin B chain ofthe fusion protein, namely [B-chain]-[X2]-[Linker]-[X3]-[A-chain], islinked with the 3′ end of a DNA containing the promoter region of anexpression system to enable the expression of the fusion proteinportion. When the fusion protein contains a leader peptide derived froma CWP, it is preferred that the leader peptide is linked to a CWP(particularly MWP) signal peptide at its 5′ end. The informationregarding MWP sequences can be found in Yamagata, H. et al., J.Bacteriol., 169:1239-1245, 1987 or Tsuboi, A. et al., J. Bacteriol.,170:935-945, 1988, and one can refer to them. The signal peptidegenerally helps to direct an expressed and translated protein to a cellmembrane and to secrete the protein extracellularly. A secreted proteinis advantageous because it can be more readily isolated and purifiedcompared to non-secreted protein.

For the fusion protein, the enzymatic cleavage of X1 includes the use ofan enzyme of which no cleavage site is contained in insulin B chain orinsulin A chain, such as factor Xa, thrombin and enterokinase. When aGly or Ser residue is attached to the N-terminus of insulin B chain, ifsuch a residue has no influence to the activity of the resultinginsulin, then TEV protease may be used. On the other hand, the chemicalcleavage of the X1may include a selective cleavage at the C-terminus ofmethionine (J. Biol. Chem., 237:1856-1860, 1962) and a selectivecleavage at the C-terminus of tryptophan (Methods in Enzymol.,91:318-324, 1983). According to a preferred embodiment of the presentinvention, all of X1 to X3 may be cleaved simultaneously, in which theenzyme used is thrombin and the amino acid sequences of X1 to X3 are asfollows: X1=GlySerLeuGlnProArg (SEQ ID NO:1); X2=ArgGlyHisArgPro (SEQ IDNO:2); and X3=ProArg. In this case, X1, X2 and X3 with other amino acidsequences may also be employed, provided that the intended cleavage withthrombin can be achieved. For example, in the above amino acidsequences, the following substitutions are possible for such cleavage:for X1 and X3, Ser=Val, Glu, Phe, Asp, Pro, Ileu, Gly, Lys, Arg, Ala,Gln, Asn or Leu; Leu=Arg, Val, Phe, Asp, Gly, Leu, His, Ileu, Met, Thror Lys; Gln=Gln, Phe, Tyr, Gly, Ileu, Asn, Ala, Arg, Thr, Ser. Leu, Valor Cys; Pro=Ala or Val; and Arg=Lys (SEQ ID NO:22; Chang, J-Y, Eur. J.Biochem., 151:217-224, 1985; Kawabata, S. et al., Eur. J. Biochem.,172:17-25, 1988); and for X2, Arg=Lys; Gly=Thr, Ileu, His, Ser, Ala,Phe, Val, Asn, Asp, Leu or Pro; His=Pro, Trp, Cys, Gln, Thr, Ser, Val,Leu, Ala, Phe or Gly; Arg =Val, Pro, Glu, Asn, Asp, Ser, Met, Lys, Ala,Gln, Gly, Trp or Thr; Pro =Val, Thr, Leu, Ser, Asp, Gly, Tyr, Ileu, Asn,Arg, His or Glu (SEQ ID NO:23; Chang, J-Y, Eur. J. Biochem.,151:217-224, 1985).

The linker comprising at least one amino acid residue is usually presentbetween functional domains in a protein and can joint the domainswithout no influence on the fuictions of the domains. enzymaticallycleavable sequence, and serves for the formation of disulfide bondsbetween insulin B chain and insulin A chain and for the readilyexpression of the fusion protein. The linker may comprise at least oneamino acid residue and any kind of amino acid(s) may be used, providedthat it exerts the same function. According to a preferred embodiment ofthe present invention, the linker may preferably comprise the C peptideof proinsulin. According to an embodiment of the present invention, thelinker comprises the following sequence:

GluAlaGluAspLeuGlnValGlyGlnValGluLeuGlyGlyGlyProGlyAlaGlySer

LeuGlnProLeuAlaLeuGluGlySerLeuGln (SEQ ID NO:3).

In the present invention, the DNA encoding the fusion protein isexpressed in the form linked with the 3′ end of a DNA containing apromoter region for an expression system employed. Examples of thepromoter include bacteriophage λpL promoter, T7 promoter, E. colitrp-lac promoter (Maniatis, T. et al., Molecular Cloning 2nd ed., ALaboratory Manual, Cold Spring Harbor Laboratory, 1989), yeast PRBIpromoter (BIO/TECHNOLOGY 9:183-187, 1991), GAPDH promoter(BIO/TECHNOLOGY 12:381-384, 1994), viral LTR promoter, SV40 promoter(Maniatis, T. et al., Molecular Cloning 2nd ed., A Laboratory Manual,Cold Spring Harbor Laboratory, 1989). According to an embodiment of thepresent invention, the fusion protein is linked with the 3′ end of aDNA-sequence containing a promoter region derived from a Bacillusbacterium. The available promoter includes, but is not limited to, MWPpromoter derived from Bacillus brevis strain 47 (JP-A-1-58950 andJP-A-7-108224), HWP promoter derived from Bacillus brevis strain HPD31(JP-A-4-278091 and JP-A-6-133782).

The DNA of the present invention may be prepared by any combination ofthe techniques known in the art. For example, constituent DNA sequencesof the DNA may be prepared separately by chemical synthesis methods orcloning methods and ligated in sequence with a ligase, and the resultingDNA sequence may be subjected to polymerase chain reaction (PCR) incombination, thereby producing the intended DNA. The details of thespecific preparation method for the DNA will be appreciated withreference to the examples below. In the preparation of the DNA,conventional techniques may be employed, such as those described inManiatis, T. et al., Molecular Cloning 2nd ed., A Laboratory Manual,Cold Spring Harbor Laboratory, 1989; and Innis, M. A. et al., PCRProtocols, A guide to methods and applications, Academic Press, 1990.

The DNA encoding human insulin comprising insulin B chain, C peptide andA chain may be obtained from a commercially available humanpancreas-derived mRNA using commercially available cDNAlst-strandsynthesis kit (Pharmacia) or the like. When short-strand DNAs (asprimers) can be synthesized based on known DNA sequences using acommercially available DNA synthesizer, the DNA fragments encoding the Bchain, the C peptide and the A chain may be amplified by a conventionalPCR method. In this case, the PCR may be performed for 20 cycles or moreunder the following conditions: DNA denaturation (e.g., at 94° C. for 30sec.-1 min.); annealing to the primer (e.g., at about 45-60° C. for 30sec.-1 min.); and elongation reaction (e.g., at 72° C. for 30 sec. ormore).

The present invention also provides a vector containing the DNA. Thevector that can be used in the present invention is needed to have atleast the following properties: it has an appropriate insertion site(i.e., a restriction site) to which the DNA of the present invention canbe inserted; it can express the DNA in a host cell; and it isautonomously replicable in the host cell. The vector generally containsa promoter, in which the promoter is operably linked with the upstreamof the DNA of interest. The vector may contain an origin of replicationand a terminator sequence, and may also contain a selectable marker suchas a drug resistance gene or an auxotrophy-complement gene. The vectorof the present invention is preferably a plasmid which is replicable ina Bacillus bacterium. Examples of the plasmid include, but are notlimited to, pNU200, pHY500 (Proc. Natl. Acad. Sci. USA, 86:3589-3593,1989), pHY4831 (J. Bacteriol., 169:1239-1245, 1987), pNU100 (Appi.Microbiol. Biotechnol., 30:75-80, 1989), pNU211 (J. Biochem.,112:488-491, 1992), pNU211R2L5 (Japanese Patent Application Laid-openNo. 7-170984), pHY700 (Japanese Patent Application Laid-open No.4-278091), pHT210 (Japanese Patent Application laid-open No. 6-133782),pHT110R2L5 (Appl. Microbiol. Biotechnol., 42:358-363, 1994). Accordingto an embodiment of the present invention, an expression vectorpNU-mPINS can be prepared by the construction process as illustrated inFIG. 3.

The present invention also provides a host cell transformed with thevector as defined above. The host cell may be prokaryotic cells (e.g.,bacteria) or prokaryotic cells (e.g., fungi, yeasts, animal cells, plantcells), and preferably a Bacillus bacterium. Examples of Bacillusbacterium as the host include, but are not limited to, Bacillus brevisstrain 47 (FERM P-7224; JP-A- 60-58074 and JP-A-62-201589), strain 47K(JP-A- 2-257876), strain 31OK (JP-A- 6-296485) and strain HPD31 (FERMBP-1087; JP-A- 4-278091). The recombinant bacterium Bacillus brevisstrain 47-5Q transformed with an expression vector pNU-mPINS has beendeposited under the terms of the Budapest Treaty on Apr. 20, 1999 at theNational Institute of Bioscience and Human-technology, Agency ofIndustrial Science and Technology, Japan (1-3, Higashi 1-chome,Tsukuba-shi, Ibaragi-ken, Japan) under the accession No. FERM BP-6706.

The expression vector prepared as mentioned above is then introducedinto a competent host cell, preferably a Bacillus bacterium cell. Thebacterium cell is cultured in a suitable culture medium under theconditions which allow the expression of the DNA to produce therecombinant fusion polypeptide of interest extracellularly orintracellularly, preferably extracellularly. The recombinant fusionpolypeptide is then collected and purified The introduction of theexpression vector into the host cell may be performed by anyconventional method such as electroporation (Methods in Enzymol.,217:23-33, 1993). The purification of the fusion polypeptide may beperformed by an appropriate combination of any conventional methods suchas solvent extraction, ultrafiltration, ammonium sulfate fractionation,HPLC, gel filtration chromatography, ion exchange chromatography,affinity chromatography, hydrophobic interaction chromatography,electrophoresis and isoelectric focusing.

The fusion polypeptide may be treated with a protease and/or a peptidasewhich can cleave the fission polypeptide enzymatically, such as thrombinand carboxypeptidase B as used in the examples below, resulting in theproduction of insulin. As illustrated in FIG. 1, at first, thrombincleaves between the leader peptide (Y) and the chain, between theB-chain and the linker and between the linker and the A chain undersuitable conditions. Preferable conditions for the specific cleavagewith thrombin are as follows: pH 7.5-8.5 (preferably with Tris buffer);temperature of 3-6° C., preferably 4° C.; the substrate:enzyme ratio=5:1to 125:1 (molar ratio), more preferably 25:1; a time period of 1-24hours. Next, carboxypeptidase B removes the Arg residue remaining at theC terminus of the B-chain, thus producing insulin (see FIG. 1). Theenzymes may be used in amounts sufficient to cause the cleavage of thefusion protein.

According to the present invention, insulin can be obtained by culturinga transformed Bacillus bacterium prepared as described above toaccumulate a fusion protein containing an insulin sequence outside thecell, and then cleaving the collected fusion protein.

The recombinant insulin prepared in this manner has the same amino acidsequence, disulfide bridges and biological activity as those of thenaturally occurring insulin and, therefore, is useful as a therapeuticmedicament for insulin-dependent diabetes mellitus.

EXAMPLES

The present invention will be illustrated in detail in the followingexamples. These examples, however, should not be construed to limit thescope of the invention.

In the preparation of a DNA encoding a fusion protein, a method wasemployed in which DNA fragments amplified by PCR reaction were ligatedby a ligation reaction with a DNA ligase. In the specification, the term“MWPsp” means a MWP-derived signal peptide, the term “MWPmp9” meansN-terminal 9 amino acid residues of the mature MWP.

EXAMPLE 1 Construction of Vector (pmPINS) HavingMWPsp-MWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain Fusion DNA IntegratedTherein

(1) Preparation of DNA fragment for MWPsp-MWPmp9

a Template DNA:

A genomic DNA extracted from Bacillus brevis strain 47-5Q by a knownmethod (Molecular Cloning 2nd ed., A Laboratory Manual, Cold SpringHarbor Laboratory, 1989):

840 ng

b. Primers:

Forward primer: 5′-GTCGTFAACAGTGTATTGCT-3′ (SEQ ID NO:7)

Reverse primer: 5′-AGCTGTAGTAGTTGCTGC-3′ (SEQ ID NO:8)

These primers were chemically synthesized based on the nucleotidesequence of MWP determined by Yamagata, H. et al. (J. Bacteriol.,169:1239-1245, 1987) and Tsuboi, A. et al. (J. Bacteriol., 170:935-945,1988) and added at the final concentration of 0.1 μM.

c. Taq DNA Polymerase:

A commercially available product (GIBCO BRL) (5U) was added.

d. Other materials:

Tris-HCl (final concentration: 20 mM, pH 8), MgCl₂ (final concentration:2.5 mM), dNTPs (dATP, dGTP, dCTP, dTIP; final concentration: 50 μM foreach) were added.

Materials “a” to “d” were mixed in a 0.5-ml tube so that the totalvolume of the reaction solution became 100 μl. PCR reaction was thenperformed according to a conventional manner (Innis, M. A. et al., PCRProtocols, A guide to methods and applications, Academic Press, 1990)under the conditions: denaturation temperature: 94° C.-1 min.; annealingtemperature: 50° C.-1 min.; DNA chain elongation temperature: 72° C.-1min.; for 30 cycles. After the PCR was completed, the reaction solutionwas concentrated with phenol, and then applied to a 0.8% agarose gel toperform electrophoresis under conventional conditions. The agarose gelwas treated using Ultrafree C3H (Millipore) to collect a PCR product(i.e., a DNA fragment for MWPsp-MWPmp9) therefrom. The collected PCRproduct was extracted with phenol, precipitated with ethanol and thendried in vacuo. The dried product was dissolved in an appropriate volumeof distilled water, and then subjected to blunting reaction using DNAblunting kit (Takara Shuzo Co., Ltd.) according to the instructions bythe manufacturer.

(2) Preparation of DNA Fragment for Proinsulin

A blunt-ended DNA fragment for proinsulin was prepared in the samemanner as mentioned in (1), except the following things.

As the template DNA, a plasmid vector having human preproinsulin DNAintegrated therein (10 ng) was used. This recombinant plasmid vector wasprepared in the following manner. Human pancreatic cDNA was synthesizedfrom a commercially available human pancreatic mRNA (CLONTECH) using 1ststrand cDNA synthesis kit (Pharmacia) according to the instructions bythe manufacturer. PCR was performed using the CDNA as a template and aforward primer: 5′-ATGGCCCTGTGGATGCGCC-3′ (SEQ ID NO:9) and a reverseprimer: 5′-CTAGTTGCAGTAGTTCTCC-3′ (SEQ ID NO:10) both synthesized basedon the nucleotide sequence of human preproinsulin gene determined byBell, G. I. et al. (Nature, 282:525-527, 1979) under the conditions:conditions: 94° C.-1 min.; 60° C.- 1 min.; 72° C.-1 min.; for 35 cycles.The PCR product, i.e., human preproinsulin DNA, was cloned into pGEM-Tvector (Promega).

As the primers, a forward primer: 5′-TTTGTGAACCAACACCTG-3′ (SEQ IDNO:11) and a reverse primer: 5′-CTAGTTGCAGTAGTTCTCC-3′ (SEQ ID NO:10)were used.

The following PCR conditions were employed: denaturation temperature:94° C.-1 min.; annealing temperature: 47° C.-1 min.; DNA chainelongation temperature: 72° C.-30 sec.; for 25 cycles).

(3) Preparation of DNA Fragment for GSLQPR-Bchain-R

A blunt-ended DNA fragment for GSLQPR-Bchain-R was prepared in the samemanner as mentioned in (1), except the following things. The resultingDNA fragment was subjected to phosphorylation reaction using T4polynucleotide kinase (Nippon Gene Co., Ltd.) according to theinstructions by the manufacturer to prepare a phosphorylated DNAfragment for GSLQPR-Bchain-R.

-   -   As the template DNA, the proinsulin PCR product prepared in (2)        (10 ng) was used.    -   As the primers, a forward primer:        5′-GGTTCCTTGCAACCTCGTTTGTGAACCAACACCTG-3′ (SEQ ID NO:12) and a        reverse primer: 5′-GCGGGTCTTGGGTGTGTA-3′ (SEQ ID NO:13) were        used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 47° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).        (4) Preparation of DNA fragment for Linker

A blunt-ended DNA fragment for Linker was prepared in the same manner asmentioned in (1), except the following things.

-   -   As the template DNA, the proinsulin PCR product prepared in (2)        (10 ng) was used.    -   As the primers, a forward primer: 5′-GAGGCAGAGGACCTGCAG-3′ (SEQ        ID NO:14) and a reverse primer: 5′-CTGCAGGGACCCCTCCAG-3′ (SEQ ID        NO:15) were used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 55° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).        (5) Preparation of DNA Fragment for GHRP-Linker

A blunt-ended DNA fragment for GHRP-Linker was prepared in the samemanner as mentioned in (4), except the following things. The resultingDNA fragment was subjected to phosphorylation reaction using T4polynucleotide kinase (Nippon Gene Co., Ltd.) according to theinstructions by the manufacturer to prepare a phosphorylated DNAfragment for GHRP-Linker.

-   -   As the template DNA,, the PCR product (the DNA fragment for        Linker) prepared in (4) (10 ng) was used.    -   As the forward primer, a primer:        5′-GGTCACCGTCCAGAGGCAGAGGACCTGCAGGTGGGG-3′ (SEQ ID NO:16) was        used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 55° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).        (6) Preparation of DNA Fragment for Achain

A blunt-ended DNA fragment for Achain was prepared in the same manner asmentioned in (1), except the following things.

-   -   As the template DNA,, the PCR product for proinsulin prepared        in (2) (10 ng) was used.    -   As the primers, a forward primer: 5′-GGCATTGTGGAACAATGCTGT-3′        (SEQ ID NO:17) and a reverse primer:        5′-CTAGTTGCAGTAGlTCTCCAGCTGGTA-3′ (SEQ ID NO: 18) were used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 55° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).        (7) Preparation of DNA Fragment for PR-Achain

A bluntended DNA fragment for PR-Achain was prepared in the same manneras mentioned in (6), except the following things. The resulting DNAfragment was subjected to phosphorylation reaction using T4polynucleotide kinase (Nippon Gene Co., Ltd.) according to theinstructions by the manufacturer to prepare a phosphorylated DNAfragment for PR-Achain.

-   -   As the template DNA,, the PCR product (i.e., the DNA fragment        for Achain) prepared in (6) (10 ng) was used.    -   As the forward primer, a primer:        5′-CCACGTGGCATTGTGGAACAATGCTGT-3′ (SEQ ID NO: 19) was used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 55° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).        (8) Preparation of Fusion DNA for MWPsp-MWPmp9-GSLQPR-Bchain-R

A blunt-ended fusion DNA fragment for MWPsp-MWPmp9-GSLQPR-Bchain-R wasprepared in the same manner as mentioned in (1), except the followingthings.

-   -   As the template DNA,, a product prepared by mixing appropriate        amounts of the DNA fragment for MWPsp-MWPmp9 prepared in (1) and        the DNA fragment for GSLQPR-Bchain-R prepared in (3) and        reacting the mixture using a DNA ligation kit (Takara Shuzo Co.,        Ltd.) at 16° C. for 30 minutes was used.    -   As the reverse primer, a primer: 5′-GCGGGTCTTGGGTGTGTA-3′ (SEQ        ID NO:13) was used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 47° C.-1        min.; DNA chain elongation temperature: 72° C.-30 sec.; for 25        cycles).

The PCR product was phosphorylated using T4 polynucleotide kinase(Nippon Gene Co., Ltd.) according to the instructions by themanufacturer. The phosphorylated PCR product was digested with Hinc 11using a DNA ligation kit (Takara Shuzo Co., Ltd.) and integrated into avector (STRATAGENE, Blue Script SK-). E. coli strain DH5α wastransformed with the vector according to a known method (MolecularCloning 2nd ed., A Laboratory Manual, Cold Spring Harbor Laboratory,1989), and the vector (i.e., the plasmid DNA) was then isolated from thetransformant. The plasmid DNA was sequenced using a forward primer (M13forward primer) or a reverse primer (M13 reverse primer) to confirm thepresence of the fusion DNA for MWPsp-MWPmp9-GSLQPR-Bchain-R. The secondround PCR was performed using a vector having the fusion DNA forMWPsp-MWPmp9-GSLQPR-Bchain-R integrated therein as a template DNA and aforward primer: 5′-GTCGTTAACAGTGTATTGCT-3′ (SEQ ID NO:7) and a reverseprimer: 5′-GCGGGTCTTGGGTGTGTA-3′ (SEQ ID NO: 13) in the same manner asmentioned above, thereby producing a blunt-ended fusion DNA forMWPsp-MWPmp9-GSLQPR-Bchain-R.

(9) Preparation of Fusion DNA for MWPs-MWPmp9-GSLQPR-Bchain-RGHRP-Linker

A blunt-ended fusion DNA for MWPsp-MWPmp9-GSLQPR-Bchain-RGHRP-Linker wasprepared in the same manner as mentioned in (g), except the followingthings.

-   -   As the template DNA, for the first round PCR, a product prepared        by mixing appropriate amounts of the fusion DNA for        MWPspMWPmp9-GSLQPR-Bchain-R prepared in (8) and the DNA fragment        for GHRP-Linker prepared in (5) and then reacting the mixture        using a DNA ligation kit (Takara Shuzo Co., Ltd.) at 16° C. for        30 minutes was used.    -   As the reverse primer, a primer: 5′-CTGCAGGGACCCCTCCAG-3′ (SEQ        ID NO:15) was used.        (10) Preparation of a Vector Having Fusion DNA for        MWPsp-MWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain Integrated        Therein

A vector having a fusion DNA forMWPsp-MWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain integrated therein(pmPINS) was prepared in the same manner as mentioned in (8), except thefollowing things.

-   -   As the template DNA, for the first round PCR, a product prepared        by mixing appropriate amounts of the fusion DNA for        MWPsp-MWPmp9-GSLQPR-Bchain-RGHRP-Linker prepared in (9) and the        DNA fragment for PR-Achain prepared in (7) and then reacting the        mixture using a DNA ligation kit (Takara Shuzo Co., Ltd.) at        16° C. for 30 minutes was used.    -   As the reverse primer for the first round PCR, a primer:        5′-CTAGTTGCAGTAGTTCTCCAGCTGGTA-3′ (SEQ ID NO: 18) was used.    -   The following PCR conditions were employed: denaturation        temperature: 94° C.-1 min.; annealing temperature: 50° C.-1        min.; DNA chain elongation temperature: 72° C.-1 min.; for 25        cycles).

EXAMPLE 2 Expression and Secretion of Fusion DNA

(1) Nucleotide Sequence of Fusion DNA and Amino Acid Sequence of FusionProtein Encoded by the fusion DNA

The nucleotide sequence of the fusion DNA prepared in Example 1 and theamino acid sequence of a fusion protein encoded by the fusion DNA areshown in FIG. 2.

(2) Expression and Secretion of Fusion DNA

The expression of a fusion protein encoded by the fusion DNA prepared inExample 1 was performed. The fusion DNA was integrated into anexpression vector as illustrated in FIG. 3.

Specifically, the vector pmPINS into which the fusion DNA was integratedwas digested with ApaL I and Hind III. The digestion product wassubjected to electrophoresis on a 0.8% agarose gel, and a gel portioncontaining the fusion DNA was excised. Appropriate amounts of theexcised fusion DNA and an expression vector for Bacillus brevis(pNU211R2L5; JP-A-7-170984) which had been digested with ApaL I and HindIII were mixed, and the mixture was reacted using a DNA ligation kit(Takara Shuzo Co., Ltd.) at 16° C. for 30 minutes, thereby the fusionDNA was integrated into the expression vector. Thus, an expressionvector pNU-mPINS having the fusion DNA integrated therein was prepared.Bacillus brevis strain 47-5 (FERM BP-1664) was transformed with theexpression vector according to a known method (Methods in Enzymol.,217:23-33, 1993), and then plated on T2 agar culture medium [polypeptone(1%), meat extract (0.5%), yeast extract (0.2%), uracil (0.1 mg/ml),glucose (1%), erythromycin (10 μg/ml), agar (1.5%); pH 7] to collect thetransformants.

The transformants were cultured in T2 culture medium (a medium havingthe same composition as T2 agar culture medium except that agar waseliminated) at 37° C. for 1 day, and plasmid DNA was isolated therefromby a known method (Molecular Cloning 2nd ed., A Laboratory Manual, ColdSpring Harbor Laboratory, 1989). The plasmid DNA was treated with ApaL Iand Hind III to confirm the presence of the fusion DNA integratedtherein. With respect to the transformant that had been confirmed thepresence of the fusion DNA integrated therein, expression and secretionof a fusion protein encoded by the integrated fusion DNA were performed.A cell suspension which had been cultured in T2 culture medium at 37° C.for 1 days was added to a culture medium [polypeptone (3%), yeastextract (0.4%), glucose (3%), MgSO₄.7H₂O (0.01%), MnSO₄. 4H₂O (0.001%),erythromycin ( 10 μg/ml); pH 8] at a ratio of 1/1000 (by volume), andthen cultured with shaking at 30° C. for 4 days.

After the cultivation, the culture medium was centrifuged at 15,000 rpmfor 2 min. to give a culture supernatant. The culture supernatant wassubjected to electrophoretic protein analysis by a known method(Laemmli, U. K., Nature, 227:680-685, 1970). That is, the culturesupernatant (18 μl) was added with buffer 1 [125 mM Tris-HCl (pH 6.8),20% glycerol, 4% SDS, 10% 2-mercaptoethanol] (2 μl), and the mixedsolution was boiled for 5 min. The mixed solution was added with buffer2 [250 mM Tris-KCl (pH 6.5), 50% glycerol, 0.5% BPB] (4 μl). Theresulting mixed solution was electrophoresed on a 15/25% SDSpolyacrylamide gel (DAIICHI PURE CHEMICALS, Co., Ltd.) (electrophoresisbuffer: 100 mM Tris, 100 mM Tricine, 0.1% SDS). After theelectrophoresis, the gel was subjected to Coomassie staining todetermine the presence of the expression and secretion of the fusionprotein. As shown in FIG. 4, for the culture medium of the cellstransformed with pNU-mPINS containing the fusion DNA, a band (markedwith an arrow) corresponding to the fusion protein was detected (lane3); whereas for the culture medium of the cells having the vectorwithout the fusion DNA, such a band was not detected (lane 2).

EXAMPLE 3 Conversion into Insulin

(I) Isolation and Purification of Fusion Protein,MWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain

Cells transformed with pNU-mPINS was cultured at 37° C. for 1 day. Analiquot (50 μl) of the cell suspension was added to a culture medium[polypeptone (3%), yeast extract (0.4%), glucose (3%), MgSO₄.7H₂O(0.01%), MnSO₄.4H2O (0.001%), erythromycin (10 μg/ml); pH g (50 ml) Themixed culture medium was charged dividedly into six 500-ml conicalflasks, and then cultured with shaking at 30° C. for 4 days. The culturemedium was centrifuged at 9,000 tpm for 20 min. The supernatant wasdialyzed against a buffer [20 mM Na—PO₄, 150 mM, pH 8] at 4° C., andthen centrifuged at 10,000 rpm for 20 min. The supematant was applied toa Ni-chelating column (Pharmacia; 5×10 cm) to elute the fusion proteinof interest with the buffer supplemented with 60 mM imidazole. Theelution fraction was added with EDTA and benzamidine (1 mM each) andkept at 4° C. Thereafter, urea (final concentration: 1 M) and cystein(final concentration: 1 mg/ml) were fluther added to the elutionfraction. The mixed solution was adjusted to pH 10.8 with 1N NaOH andstirred at the same temperature for 1 hour. The solution was dialyzedagainst a buffer [20 mM Tris, 1 mM EDTA; pH 8.0]. The dialysate wasadded with urea (final concentration: 1 M) and 2-propanol (finalconcentration: 20%) and then applied to Q-Sepharose XL column(Pharmacia; 1.6×10 cm). The column was sufficiently equilibrated with abuffer [20 mM Tris, 1mM EDTA, 1 M urea, 20% 2-propanol; pH 8] and theneluted with the buffer supplemented under gradient with 1 M NaCl. InFIG. 5, the elution pattern is shown. The elution fractions eluted with160 mM-200 mM NaCl (shown by the arrow) were combined, and adjusted topH 3 with 1N HCl. The solution was concentrated using an ultrafilter(fractionation molecular weight: 3,000), and then applied toVydac214TP54 (CYPRESS; C4 column, 4.6×250 rnm) for purification by HPLC.The column was equilibrated with 25% acetonitrile and 0.1% TFE solution,and then eluted under gradient with 33% acetonitrile and 0.1% TFEsolution. In FIG. 6, the elution pattern is shown. The fractions elutedwith 30-31% acetonitrile (shown by the arrow) were centrifuged andconcentrated to dryness. The resulting product was used in thesubsequent cleavage experiment.

(2) Conversion Into Insulin and Purification Thereof

The fusion protein prepared in (1) as a dry product,MWPmp9-GSLQPR-Bchain-RGHRP-Linker-PR-Achain, was dissolved in aappropriate amount of 0.1% TFA, and then added with 0.1 M Tris buffer(pH 8) to the final concentration of 20 nmol/ml. The resulting solutionwas cooled to 4° C., and then added with a thrombin solution (250μmol/ml) at the substrate:enzyme ratio of 25:1 (molar base). After 9hours, a appropriate amount of 10% TFA was added to the reactionsolution to adjust to pH 2, thereby terminating the reaction. Thethrombin used was thrombin of JP grade (ITOHAM FOODS INC.) which hadbeen re-purified using Macro Prep CM (Bio Rad) and Lysine Sepharose 4B(Pharmacia).

To isolate insulin-Arg, which had an Arg residue at the C terminus ofthe B chain cleaved with thrombin, by reversed phase HPLC, thereaction-terminated solution after the thrombin treatment was applied toMightysil RP4 column (Cica-MERCK; 20×250 mm), the column wasequilibrated with 25% acetonitrile and 0.1% TFA solution and then elutedunder gradient with 35% acetonitrile and 0.1% TFA solution. Thefractions eluted with 30-31% acetonitrile were centrifuged andconcentrated to dryness. The resulting dry product was used in thesubsequent experiment.

The dry product insulin-Arg was dissolved in a appropriate amount of0.1% TFA and then added with 0.1M Tris buffer (pH 8) to the finalconcentration of 1 mg/mi. The resulting solution was added with acarboxypeptidase B solution (Sigma; 4.7 mg/ml) at the substrate:enzymeratio of 500:1 (molar base), and the reaction solution was allowed toreact at 25° C. for 12 hours. The reaction solution was added with aappropriate amount of 10% TFA to adjust to pH 2, thereby terminating thereaction. To isolate insulin from the reaction-terminated solution, thereversed phase HPLC was performed in the same manner as mentioned forthe isolation of the insulin-Arg. (3) Amino Acid Analysis of Insulin

At first, the total amino acid of the insulin was analyzed. The insulinobtained in (2) (about 2 nmoles) was added with 6N HCl (200 μl) and 5%phenol (20 μl). The resulting reaction solution was deaerated and thetube containing the reaction solution was sealed. The reaction solutionwas allowed to react at 110° C. for 24 hours to cause hydrolysisreaction, and then dried. The resulting dry product was dissolved in0.01N HCl (100 μl) and filtrated on a 0.2-μm filter. An aliquot (50 μl)of the filtrate was analyzed using Hitachi amino acid analyzer ModelL-8500 (HITACHI, Ltd.).

Next, the cysteic acid content of the insulin was analyzed. The insulinobtained in (2) (about 2 nmoles) was dissolved in a formic acid/methanol(5:1) mixed solution (40 μl), and cooled to −20° C. The cooled solutionwas added with a 99% formic acid/30% aqueous hydrogen peroxide (19:1)mixed solution (400 μl) which had been cooled to −20° C., and thenallowed to react at −20° C. for 4 hours. After the reaction wascompleted, distilled water (3 ml) was added to the reaction solution andlyophilized. The resulting dry product was hydrolyzed in the same manneras mentioned above, and then analyzed.

The analytical values for Val determined in the total amino acidanalysis and the cysteic acid analysis were compared, and the analyticalvalue for cysteic acid was converted into the analytical value forcysteine of the total amino acid analysis. As shown in Table 1, theamino acid ratio of the insulin of the present invention was almostconsistent with that of the naturally occurring insulin.

TABLE 1 INS analysis Analytical INS calculated value Amino value Numberof Residue Number of acid nmole mol % residue nmole Residue mol % Cys-5.697 12.63% 6.41 0.950 SO3H Asp 2.726 6.12% 3.11 0.921 3 5.88% Thr2.696 5.97% 3.03 0.899 3 5.88% Ser 2.437 5.40% 2.74 0.812 3 5.88% Glu6.271 13.90% 7.05 0.895 7 13.73% Pro 0.985 2.18% 1.11 0.985 1 1.96% Gly3.392 7.52% 3.81 0.848 4 7.84% Ala 1.000 2.22% 1.12 1.000 1 1.96% Cys1/26 11.76% Val* 3.205 7.10% 3.60 0.801 4 7.84% Met Ile 1.409 3.12% 1.580.705 2 3.92% Leu 5.462 12.10% 6.14 0.910 6 11.76% Tyr 3.507 7.77% 3.940.877 4 7.84% Phe 2.640 5.85% 2.97 0.880 3 5.88% Lys 0.913 2.02% 1.030.913 1 1.96% His 1.822 4.04% 2.05 0.911 2 3.92% Trp Arg 0.924 2.05%1.04 0.924 1 1.96% 45.122 100.00% 50.73 0.889 51 100.00%(4) Peptide Mapping of Insulin

The insulin obtained in (2) (hereinafter, referred to as “ITOHAMinsulin”) and a commercially available insulin, Novolin 40, (NovoNordisk Pharma)(hereinafter, referred to as “Novolin”) (5 nmoles each)were separately dissolved in 0.81 M ammonium hydrogen carbonate (50μl)and 2 mM EDTA solution (pH 7.8; 50 μl). The resulting solution wasadded with an aqueous V8 protease solution (Wako Pure ChemicalIndustries, Ltd.; 2 μml) (1.35 .92). The reaction solution was allowedto react at 25° C. for 24 hours, and 1% TFA solution was added theretoto adjust to pH 2, thereby terminating the reaction. Thereaction-terminated solution was applied to Vydac218TP54 column (4.6×250mm; C18 column), equilibrated with 5% acetonitrile and 0.1% TFAsolution, and then eluted under gradient with 35% acetonitrile and 0.1%TFA solution. In FIG. 7, the elution pattern is shown. ITOHAM insulinand Novolin showed similar elution patterns to each other. Therefore, itis concluded that the both kinds of insulin have the similar disulfidebridging forms.

EXAMPLE 4 Biological Activity of Insulin

Novolin (1.2 ml) was treated with the same manner as in Example 3 (2)using Wakocil-II 5C18 AR Prep (Wako Pure Chemical Industries, Ltd.;20×250 mm) to give insulin fractions. The insulin fractions of ITOHAMinsulin obtained in Example 3 (2) and the insulin fractions of Novolinwere analyzed using Vydac218TP54 (4.6×250 mm; C18 column). For eachinsulin sample, an aliquot was taken from the fractions so that theinsulin contents for both kinds of insulin calculated based on the mainpeak areas became same, and then dried. As shown in FIG. 8, the elutionpattern of Novolin had sub-peaks which were assumed to indicate thepresence of polymeric materials. The subpeak level (i.e., total sub-peakarea) of Novolin was 1.23 times greater than that of IHOHAM insulin.

ITOHAM insulin and Novolin thus obtained were separately dissolved in asolution containing 0.1% BSA, 0.9% NaCl and 0.1% phenol solution at thefinal concentration of 1 unit/ml (calculated based on the assumptionthat each insulin was 26 units/mg). The resulting solution (0.5 ml) wassubcutaneously injected to the back of a Japanese Wister rabbit (Kbs:JW,20.-2.5 kg). After the injection, blood was collected from the anteriorauricular vein of the rabbit over a period of time. Each blood sample(0.45 ml) was added with a mix of glycolysis-inhibiting agents (NaF:12.5 mg/ml, heparin-Na: 125 units/ml, EDTA-2Na: 48 mg/ml) (0.05 nm]) andfully mixed. The mixed solution was centrifuged at 3,000 rpm for 15 min.at 5° C., and the supernatant was used as a plasma sample. For eachplasma sample, the plasma glucose level was determined using abiochemical automated analyzer (CIBA-CORNING; Express PLUS), and theplasma insulin level was determined using EIA kit (Wako Pure ChemicalIndustries, Ltd.). The time course of the plasma glucose level and thetime course of the plasma insulin level are shown in FIG. 9 and FIG. 10,respectively. Both ITOHAM insulin and Novolin showed the decrease inplasma glucose level after the injection of insulin, demonstrating thatboth kinds of insulin acted to lower blood sugar. With respect to theplasma insulin level, both kinds of insulin showed the similar courses.The slight lower plasma glucose level and the slight higher plasmainsulin level of Novolin than those of ITOHAM insulin are considered tobe resulted from the increment of the sub-peaks in Novolin which wereconfirmed by the analysis by HPLC above.

As mentioned above, according to the present invention, a novel fusionprotein convertible into insulin can be expressed and secreted in a highlevel in a Bacillus expression system. By treating the fusion protein ofthe present invention with thrombin and carboxypeptidase B, insulinhaving the same amino acid composition and biological activity asnaturally occurring insulin can be obtained.

1. A DNA encoding a fusion protein of formula (I):[Y]-[X1]-[B-chain]-[X2]-[Linker]-[X3]-[A-chain]  (I) wherein Y is aleader peptide sequence for expression and secretion of the protein,comprising the N-terminal 9 amino acid residues of the middle wallprotein (MWP) which is one of the cell wall proteins (CWPs) from abacterium belonging to the genus Bacillus; X1 is an amino acid sequencewhich is cleavable with thrombin; B-chain is the amino acid sequence ofinsulin B chain; X2 is an amino acid sequence which is cleavable withthrombin Linker is a linker sequence comprising at least one amino acidresidue; X3 is an amino acid sequence which is cleavable with thrombin;and A-chain is the amino acid sequence of insulin A chain, and whereinthe Y, X1, B-chain, X2, Linker, X3 and A-chain are ligated in the orderindicated in formula (1).
 2. The DNA of claim 1, wherein the amino acidsequences X1, X2 and or X3, which are used for enzymatic cleavage of afusion protein, consist of the following sequences:X1=GlySerLeuGlnProArg (SEQ ID NO:1); X2=ArgGlyHisArgPro (SEQ ID NO:2);and X3=ProArg.
 3. The DNA of claim 1, wherein the linker sequencecomprises the following amino acid sequence:GluAlaGluAspLeuGlnValGlyGlnValGluLeuGlyGlyGlyProGlyAlaGlySerLeuGlnProLeuAlaLeuGluGlySerLeuGln (SEQ ID NO:3).
 4. The DNA of claim1, wherein the DNA comprises a CWP signal peptide attached at the 5′ endof the DNA.
 5. A DNA comprising a nucleotide sequence encoding an aminoacid sequence shown in SEQ ID NO:21.
 6. The DNA of claim 5, wherein theDNA comprises a nucleotide sequence shown in SEQ ID NO:20.
 7. A DNAcomprising a DNA sequence which comprises a promoter region required forthe expression of a recombinant protein in a prokaryote or eukaryote,the DNA sequence being attached at the 5′ end of the DNA of claim
 1. 8.The DNA of claim 7, wherein the DNA sequence which comprises a promoterregion is derived from a bacterium belonging to the genus Bacillus. 9.The DNA of claim 8, wherein the DNA sequence which comprises a promoterregion is derived from the CWP from a bacterium belonging to the genusBacillus.
 10. A vector containing the DNA of claim
 7. 11. A host celltransformed with the vector of claim
 10. 12. A bacterium belonging tothe genus Bacillus which is transformed with the vector of claim
 10. 13.The bacterium of claim 12, wherein the bacterium is Bacillus brevis. 14.A method for producing insulin, wherein the method comprises: culturingthe host cell of claim 11 or the bacterium of claim 12, in a culturemedium, to express a fusion protein encoded by a DNA of interest in thehost cell or bacterium; collecting the fusion protein; and subjectingthe fusion protein to an enzymatic cleavage treatment to isolateinsulin.
 15. The method of claim 14, wherein the fusion proteincomprises an amino acid sequence shown in SEQ ID NO:21.
 16. The methodof claim 14, wherein the expressed fusion protein is separated andpurified from the host cell or bacterium, or from the cultured medium.17. The method of claim 14, wherein the enzymatic cleavage treatment isperformed with thrombin or carboxypeptidase B.
 18. A fusion proteincomprising an amino acid sequence shown in SEQ ID NO:21.